Modified and stabilized gdf propeptides and uses thereof

ABSTRACT

Modified and stabilized propeptides of Growth Differentiation Factor proteins, such as GDF-8 and Bone Morphogenetic Protein-11, are disclosed. Also disclosed are methods for making and using the modified propeptides to prevent or treat human or animal disorders in which an increase in muscle tissue would be therapeutically beneficial. Such disorders include muscle or neuromuscular disorders (such as amyotrophic lateral sclerosis, muscular dystrophy, muscle atrophy, congestive obstructive pulmonary disease, muscle wasting syndrome, sarcopenia, or cachexia), metabolic diseases or disorders (such as such as type 2 diabetes, noninsulin-dependent diabetes mellitus, hyperglycemia, or obesity), adipose tissue disorders (such as obesity), and bone degenerative diseases (such as osteoporosis).

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 10/071,499,filed Feb. 8, 2002, which claims the benefit of U.S. provisionalapplication Ser. No. 60/267,509, filed on Feb. 8, 2001, the entiredisclosure of which is hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to inhibitors of Growth Differentiation Factor-8(GDF-8) proteins and methods for their use. More particularly, theinvention provides modified and stabilized propeptides of GDF-8 proteinswhich inhibit the activity of GDF-8. The invention is particularlyuseful for preventing or treating human or animal disorders in which anincrease in skeletal muscle tissue would be therapeutically beneficial.Such disorders include muscle or neuromuscular disorders (such asamyotrophic lateral sclerosis, muscular dystrophy, muscle atrophy,congestive obstructive pulmonary disease, muscle wasting syndrome,sarcopenia, or cachexia), metabolic diseases or disorders (such as type2 diabetes, noninsulin-dependent diabetes mellitus, hyperglycemia, orobesity), adipose tissue disorders (such as obesity), or bonedegenerative diseases (such as osteoporosis).

BACKGROUND OF THE INVENTION

Growth and Differentiation Factor-8 (GDF-8), also known as myostatin, isa member of the Transforming Growth Factor-beta (TGF-β) superfamily ofstructurally related growth factors, all of which possess importantgrowth-regulatory and morphogenetic properties (Kingsley et al. (1994)Genes Dev. 8:133-46; Hoodless et al. (1998) Curr. Topics Microbiol.Immunol. 228:235-72). GDF-8 is a negative regulator of skeletal musclemass, and there is considerable interest in identifying factors whichregulate its biological activity. For example, GDF-8 is highly expressedin the developing and adult skeletal muscle. The GDF-8 null mutation intransgenic mice is characterized by a marked hypertrophy and hyperplasiaof the skeletal muscle (McPherron et al. (1997) Nature 387:83-90).Similar increases in skeletal muscle mass are evident in naturallyoccurring mutations of GDF-8 in cattle (Ashmore et al. (1974) Growth38:501-507; Swatland and Kieffer (1994) J. Anim. Sci. 38:752-757;McPherron and Lee (1997) Proc. Natl. Acad. Sci. U.S.A. 94:12457-12461;and Kambadur et al. (1997) Genome Res. 7:910-915). Recent studies havealso shown that muscle wasting associated with HIV-infection in humansis accompanied by increases in GDF-8 protein expression(Gonzalez-Cadavid et al. (1998) PNAS 95:14938-43). In addition, GDF-8can modulate the production of muscle-specific enzymes (e.g., creatinekinase) and modulate myoblast cell proliferation (WO 00/43781).

In addition to its growth-regulatory and morphogenetic properties inskeletal muscle, GDF-8 may also be involved in a number of otherphysiological processes (e.g., glucose homeostasis), as well as abnormalconditions, such as in the development of type 2 diabetes and adiposetissue disorders, such as obesity. For example, GDF-8 modulatespreadipocyte differentiation to adipocytes (Kim et al. (2001) B.B.R.C.281:902-906).

Like TGF-β-1, -2, and -3, the GDF-8 protein is synthesized as aprecursor protein consisting of an amino-terminal propeptide and acarboxy-terminal mature domain (McPherron and Lee, 1997, supra) as wellas a signal sequence which directs the protein to the extracellulardomain and is also cleaved from the protein. It is believed that beforecleavage of the propeptide, the precursor GDF-8 protein forms ahomodimer. The amino-terminal propeptide is then cleaved from the maturedomain and the cleaved propeptide may remain noncovalently bound to themature domain dimer, inhibiting its biological activity (Miyazono et al.(1988) J. Biol. Chem. 263:6407-6415; Wakefield et al. (1988) J. Biol.Chem. 263:7646-7654; and Brown et al. (1990) Growth Factors 3:35-43). Itis believed that two GDF-8 propeptides bind to the GDF-8 mature dimer(Thies et al. (2001) Growth Factors 18:251-259). Due to thisinactivating property, the propeptide is known as the“latency-associated peptide” (LAP), and the complex of mature domain andpropeptide is commonly referred to as the “small latent complex” (Gentryand Nash (1990) Biochemistry 29:6851-6857; Derynck et al. (1995) Nature316:701-705; and Massague (1990) Ann. Rev. Cell Biol. 12:597-641). Themature domain is believed to be active as a homodimer when thepropeptide is removed. Other proteins are also known to bind to GDF-8 orstructurally related proteins and inhibit their biological activity.Such inhibitory proteins include follistatin (Gamer et al. (1999) Dev.Biol 208:222-232) and follistatin-related proteins.

Further, a number of human and animal disorders are associated with lossof or functionally impaired muscle tissue, including muscular dystrophy,muscle atrophy, congestive obstructive pulmonary disease, muscle wastingsyndrome, sarcopenia, and cachexia. To date, very few reliable oreffective therapies exist for these disorders. The terrible symptomsassociated with these disorders could be substantially reduced byemploying therapies that increase the amount of muscle tissue inpatients suffering from the disorders. Such therapies wouldsignificantly improve the quality of life for these patients and couldameliorate many effects of these diseases. Thus, there is a need in theart to identify new therapies that contribute to an overall increase inmuscle tissue in patients suffering from these disorders.

The present invention fills this need by providing modified andstabilized GDF propeptides that retain their biological activity andinhibit the activity of GDF proteins. The modified propeptides of theinvention may be used to treat human or animal disorders in which anincrease in muscle tissue would be therapeutically beneficial.

SUMMARY OF THE INVENTION

GDF-8 is involved in the regulation of many critical biologicalprocesses. Due to its key function in these processes, GDF-8 may be adesirable target for therapeutic intervention. Although naturallyoccurring GDF-8 propeptide may be an attractive means of GDF modulationfrom an efficacy and toxicity perspective, the present inventors havediscovered that the circulatory half-life of the natural propeptide maybe too short for the molecule to have practical therapeutic orprophylactic utility.

Accordingly, the present invention is based, at least in part, on thediscovery that the propeptide of Growth Differentiation Factor-8 (GDF-8)inhibits the activity of GDF-8 protein, and that other TransformingGrowth Factor-beta (TGF-β) proteins which are related in structure toGDF-8, such as Bone Morphogenetic Protein-11 (BMP-11; also known asGDF-11), are also inhibited by GDF-8 propeptide. The present inventionthus provides compositions and methods for inhibiting GDF proteins, aswell as methods for identifying, making and using such inhibitors.

As noted above, the present invention is also based, in part, on thediscovery that the natural GDF-8 propeptide has a relatively short invivo half-life, which may prevent practical therapeutic or prophylacticutility. Thus, the present invention provides modified GDF-8 propeptidesand modified BMP-11 propeptides having improved pharmacokineticproperties, such as increased circulatory half-life or increasedprotection from proteolytic degradation.

The presently disclosed GDF-8 propeptides or BMP-11 propeptides may bestabilized by any means known in the art. For example, in oneembodiment, the modified propeptide is a fusion protein comprising aGDF-8 propeptide and the Fc region of an IgG molecule. GDF-8 propeptidefusion proteins may comprise, as the active subunit, the propeptide of aGDF-8 protein, or an active portion of the GDF-8 propeptide, fused to anFc region of an IgG molecule. In other embodiments, or in addition, theGDF-8 propeptide may be glycosylated, or linked to albumin or anonproteineous polymer.

In other embodiments, the modified propeptide is a fusion proteincomprising a BMP-11 propeptide and the Fc region of an IgG molecule.BMP-11 propeptide fusion proteins may comprise, as the active subunit,the propeptide of a BMP-11 protein, or an active portion of the BMP-11propeptide, fused to an Fc region of an IgG molecule. In otherembodiments, or in addition, the BMP-11 propeptide may be glycosylated,or linked to albumin or a nonproteineous polymer.

The modified GDF-8 propeptides or modified BMP-11 propeptides of theinvention are capable of inhibiting the activity, expression,processing, or secretion of a GDF-8 protein, mature GDF-8, or a GDF-8homodimer or other active GDF-8 molecule. The modified GDF-8 propeptidesor modified BMP-11 propeptides of the invention may be administered to apatient, in a therapeutically effective dose, to treat or preventmedical conditions in which an increase in muscle tissue would betherapeutically beneficial. Diseases and disorders that may be treatedby the modified GDF-8 propeptides or modified BMP-11 propeptides includebut are not limited to muscle or neuromuscular disorders (such asamyotrophic lateral sclerosis, muscular dystrophy, muscle atrophy,congestive obstructive pulmonary disease, muscle wasting syndrome,sarcopenia, or cachexia), metabolic diseases or disorders (such as suchas type 2 diabetes, noninsulin-dependent diabetes mellitus,hyperglycemia, or obesity), adipose tissue disorders (such as obesity),and bone degenerative diseases (such as osteoporosis).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relative biological activities of GDF-8, BMP-11 andactivin in a reporter gene assay.

FIG. 2 shows the binding properties of purified biotinylated human GDF-8in an ActRIIB binding assay.

FIG. 3 shows induction of pGL3(CAGA)₁₂ reporter activity at the ED₅₀ forGDF-8, BMP-11, and activin in the presence of GDF-8 propeptide.

FIG. 4 shows the dose-dependent inhibition of biotinylated GDF-8 bindingto ActRIIB by GDF-8 propeptide.

FIG. 5 shows the binding of GDF-8 to L6 cells.

FIG. 6 shows the dose-dependent inhibition of GDF-8 specific binding toL6 cells by GDF-8 propeptide.

FIG. 7A shows the amino acid sequence of a murine GDF-8 propeptide-Fcfusion protein, as provided in SEQ ID NO:20.

FIG. 7B shows a murine GDF-8 propeptide-Fc fusion protein with a shortglycine-serine-glycine-serine (GSGS, SEQ ID NO:17) linker separating theGDF-8 propeptide from the Fc region, as provided in SEQ ID NO:21.

FIG. 8A shows the effects of human GDF-8 propeptide and two murine GDF-8propeptide-Fc fusion proteins on GDF-8 binding in an ActRIIB competitionELISA.

FIG. 8B is a table comparing the IC₅₀'s of human GDF-8 propeptide andtwo murine GDF-8 propeptide-Fc fusion proteins.

FIG. 9 shows the relative biological activities of human GDF-8propeptide and murine GDF-8 propeptide-Fc fusion protein in a reportergene assay.

FIG. 10 shows the pharmacokinetics of iodinated GDF-8 propeptide and aGDF-8 propeptide-Fc fusion protein after a single intravenousadministration of 0.2 μg/mouse or 2 μg/mouse, respectively.

FIG. 11A shows the amino acid sequence of a human GDF-8 propeptide-IgG1Fc fusion protein, as provided in SEQ ID NO:22.

FIG. 11B shows the amino acid sequence of a human GDF-8 propeptide-IgG1Fc fusion protein modified for reduced effector function, as provided inSEQ ID NO:23.

FIG. 12 shows the dose-dependent inhibition of GDF-8 binding in anActRIIB competition ELISA by human GDF-8 propeptide and two human GDF-8propeptide-Fc fusion proteins.

FIG. 13 is a graph showing that modified GDF-8 propeptide administeredin vivo increases gastrocnemius and quadricep mass, and decreases fatmass, but does not effect kidney or liver mass compared to untreatedmice or mice treated with the control protein, IgG2aFc.

FIGS. 14A-P illustrate the amino acid and nucleic acid sequencescorresponding to SEQ ID NO.s 1-16 and annotations thereto.

Definitions

The terms “GDF-8”, “GDF-8 protein”, or “GDF-8 polypeptide” refer to aspecific growth and differentiation factor including but not limited tothat set forth in SEQ ID NO:1, and any now known or later describedhomologs thereof, including homologs of other species. Particularlypreferred are mammalian homologs, most preferably human. The presentinvention also encompasses GDF-8 molecules and homologs from all othersources, including but not limited to GDF-8 from bovine, dog, cat,chicken, murine, rat, porcine, ovine, turkey, baboon, and fish. VariousGDF-8 molecules have been described in McPherron et al. (1997) Proc.Natl. Acad. Sci. USA 94:12457-12461. Homologs are well known in the art.Homology may be determined by any method known in the art, or asdescribed herein. The GDF-8 or GDF-8, protein may be naturally occurringor synthetic. These terms include the full-length unprocessed precursorform of the protein (“GDF-8 precursor protein”), as well as the matureforms resulting from post-translational cleavage of the propeptide. Theterms also refer to any fragments or variants of GDF-8 that maintain oneor more biological activities associated with a GDF-8 protein, asdiscussed herein, including sequences that have been modified withconservative or non-conservative changes to the amino acid sequence.

“Mature GDF-8” refers to the protein or polypeptide that is cleaved fromthe carboxy-terminal domain of the GDF-8 precursor protein. A human formof mature GDF-8 protein is provided in SEQ ID NO:3. The mature GDF-8 maybe naturally occurring or synthetic. The mature GDF-8 may be present asa monomer, homodimer, or may be present in a GDF-8 latent complex.Depending on the in vivo or in vitro conditions, mature GDF-8 mayestablish an equilibrium among any or all of these different forms.Without limitation as to the mechanism, mature GDF-8 is believed to bebiologically active as homodimer. In its biologically active form, themature GDF-8 is also referred to as “active GDF-8.”

The phrase “GDF-8 activity” refers to one or more of physiologicallygrowth-regulatory or morphogenetic activities associated with activeGDF-8 protein. For example, active GDF-8 is a negative regulator ofskeletal muscle mass. Active GDF-8 can also modulate the production ofmuscle-specific enzymes (e.g., creatine kinase), stimulate myoblast cellproliferation, and modulate preadipocyte differentiation to adipocytes.GDF-8 also believed to increase sensitivity to insulin and glucoseuptake in peripherial tissues, particularly in skeletal muscle oradipocytes. Accordingly, GDF-8 biological activities include but are notlimited to inhibition of muscle formation, inhibition of muscle cellgrowth, inhibition of muscle development, decrease in muscle mass,regulation of muscle-specific enzymes, inhibition of myoblast cellproliferation, modulation of preadipocyte differentiation to adipocytes,increasing sensitivity to insulin, regulations of glucose uptake,glucose hemostasis, and modulate neuronal cell development andmaintenance. A human form of the full length GDF-8 precursor protein isprovided in SEQ ID NO:1.

“GDF-8 propeptide” refers to a polypeptide that is cleaved from theamino-terminal domain of the GDF-8 precursor protein. The GDF-8propeptide is associated with one or more biological activitiesincluding but not limited to the ability to bind to a mature GDF-8protein or homodimer, the ability to inhibit one or more GDF-8biological activities, the ability to enhance muscle development, theability to enhance muscle mass, the ability to promote muscle formation,and the ability to promote muscle cell proliferation. The GDF-8propeptide may be naturally occurring or synthetic. An example of aGDF-8 propeptide includes but is not limited to a human form of GDF-8propeptide provided in SEQ ID NO:5. In one embodiment, the GDF-8propeptide is capable of binding to the propeptide binding domain ofmature GDF-8. In another embodiment, the GDF-8 propeptide is capable ofinhibiting one or more activities of a mature GDF-8.

The term “GDF-8 inhibitor” includes any agent capable of inhibiting theactivity, expression, processing, or secretion of a GDF-8 protein,mature GDF-8, or a GDF-8 homodimer or other active GDF-8 moleculeincluding but not limited to modified GDF-8 propeptides and modifiedBMP-11 propeptides. A GDF-8 inhibitor also includes any agent capable ofenhancing the binding of a GDF-8 propeptide to a mature GDF-8 molecule,or to a GDF-8 homodimer. Such inhibitors include but are not limited toproteins, antibodies, peptides, peptidomimetics, ribozymes, anti-senseoligonucleotides, double-stranded RNA, and other small molecules. In oneembodiment, the activity of a GDF-8 protein is inhibited by a modifiedGDF-8 propeptide as described in Examples 3-6. In another embodiment,the activity of a GDF-8 protein is inhibited by a modified BMP-11propeptide.

“GDF-8 latent complex” refers to a complex of proteins formed between amature GDF-8 homodimer and a GDF-8 propeptide. Without limitation as tomechanism it is believed that two GDF-8 propeptides associate with twomolecules of mature GDF-8 in the homodimer to form an inactivetetrameric or latent complex. The latent complex may include other GDFinhibitors in place of or in addition to one or more of the GDF-8propeptides.

The term “modified GDF-8 propeptide” refers to a GDF-8 inhibitor whichcomprises a modified GDF-8 propeptide, fragment or variant thereof whichretains one or more biological activities of a GDF-8 propeptide andfurther comprises a stabilizing modification as set forth herein.Variant forms of GDF-8 propeptide, include, but are not limited to, forexample, GDF-8 propeptides that have been modified to include mutations(including insertion, deletion, and substitution of amino acids) in thesignal peptide or propeptide proteolytic cleavage sites to make thesites less susceptible to proteolytic cleavage. In a preferredembodiment, the modified GDF-8 propeptide has a substantially increasedhalf life relative to that of the corresponding unmodified GDF-8propeptide. In a highly preferred embodiment, the modified GDF-8propeptide of the invention has an increased in vivo half life (asmeasured, for example, by the method set forth in Example 8). In anotherembodiment, the modified GDF-8 propeptide is capable of inhibiting oneor more activities of a mature GDF-8.

The terms “BMP-11”, “BMP-11 protein”, or “BMP-11 polypeptide” refer to aspecific growth and differentiation factor including but not limited tothat set forth in SEQ ID NO:7, and any now known or later describedhomologs thereof, including homologs of other species. Particularlypreferred are mammalian homologs, most preferably human. The presentinvention also encompasses BMP-11 molecules and homologs from all othersources, including but not limited to BMP-11 from bovine, dog, cat,chicken, murine, rat, porcine, ovine, turkey, baboon, and fish. VariousBMP-11 molecules have been described in McPherron et al. (1997) Proc.Natl. Acad. Sci. USA 94:12457-12461. Homologs are well known in the art.Homology may be determined by any method known in the art, or asdescribed herein. The BMP-11 or BMP-11 protein may be naturallyoccurring or synthetic. These terms include the full-length unprocessedprecursor form of the protein (“BMP-11 precursor protein”), as well asthe mature forms resulting from post-translational cleavage of thepropeptide. The terms also refer to any fragments or variants of BMP-11that maintain one or more biological activities associated with a BMP-11protein, as discussed herein, including sequences that have beenmodified with conservative or non-conservative changes to the amino acidsequence.

“Mature BMP-11” refers to the protein or polypeptide that is cleavedfrom the carboxy-terminal domain of the BMP-11 precursor protein. Ahuman form of mature BMP-11 protein is provided in SEQ ID NO:9. Themature BMP-11 may be naturally occurring or synthetic. The mature BMP-11may be present as a monomer, homodimer, or may be present in a BMP-11latent complex. Depending on the in vivo or in vitro conditions, matureBMP-11 may establish an equilibrium among any or all of these differentforms. Without limitation as to the mechanism, mature BMP-11 is believedto be biologically active as homodimer. In its biologically active form,the mature BMP-11 is also referred to as “active BMP-11.”

“BMP-11 propeptide” refers to a polypeptide that is cleaved from theamino-terminal domain of the BMP-11 precursor protein. The BMP-11propeptide is associated with one or more biological activitiesincluding but not limited to the ability to bind to a mature GDF-8protein or homodimer, the ability to inhibit one or more GDF-8biological activities, the ability to enhance muscle development, theability to enhance muscle mass, the ability to promote muscle formation,and the ability to promote muscle cell proliferation. The BMP-11propeptide may be naturally occurring or synthetic. An example of aBMP-11 propeptide includes but is not limited to a human form of BMP-11provided in SEQ ID NO:11. In one embodiment, the BMP-11 propeptide iscapable of binding to the propeptide binding domain of mature GDF-8. Inanother embodiment, the BMP-11 propeptide is capable of inhibiting oneor more activities of a mature GDF-8.

The term “modified BMP-11 propeptide” refers to a GDF-8 inhibitor whichcomprises a modified BMP-11 propeptide, or fragment or variant thereof,which retains one or more biological activities of a BMP-11 propeptideand further comprises a stabilizing modification as set forth herein.Variant forms of BMP-11 propeptide, include, but are not limited to, forexample, BMP-11 propeptides that have been modified to include mutations(including insertion, deletion, and substitution of amino acids) in thesignal peptide or propeptide proteolytic cleavage sites to make thesites more or less susceptible to proteolytic cleavage. In a preferredembodiment, the BMP-11 propeptide inhibitor is modified to provide anincreased in vivo half life (measured in one embodiment in Example 8)relative to that of the corresponding unmodified BMP-11 propeptide. Inanother embodiment, the modified BMP-11 propeptide is capable ofinhibiting one or more activities of a mature GDF-8.

The GDF-8 propeptides of the invention and BMP-11 propeptides of theinvention are collectively referred to as “GDF propeptides.”

The GDF-8 proteins of the invention and the BMP-11 proteins of theinvention are collectively referred to as “GDF polypeptides” or “GDFproteins.”

The methods and compositions of the invention provide GDF inhibitorswhich reduce the activity of GDF protein relative to the activity of aGDF protein as compared to the same GDF protein not bound by aninhibitor. In certain embodiments, the activity of a GDF protein, whenbound by one or more of the modified GDF propeptides is reduced at leastabout 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55%, preferably atleast about 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%,84%, 86%, or 88%, more preferably at least about 90%, 91%, 92%, 93%, or94%, and even more preferably at least about 95% to 100% relative to thesame GDF protein that is not bound by said modified propeptides. In onepreferred embodiment, the GDF inhibitor is a modified GDF-8 propeptideor a modified BMP-11 propeptide covalently linked to an Fc region of anIgG molecule.

The term “stabilizing modification” is any modification known in the artor set forth herein capable of stabilizing a protein, enhancing the invitro half life of a protein, enhancing circulatory half life of aprotein and/or reducing proteolytic degradation of a protein. Suchstabilizing modifications include but are not limited to fusion proteins(including, for example, fusion proteins comprising a GDF propeptide anda second protein), modification of a glycosylation site (including, forexample, addition of a glycosylation site to a GDF propeptide), andmodification of carbohydrate moiety (including, for example, removal ofcarbohydrate moieties from a GDF propeptide). In the case of astabilizing modification which comprises a fusion protein (e.g., suchthat a second protein is fused to a GDF propeptide), the second proteinmay be referred to as a “stabilizer portion” or “stabilizer protein. Forexample, in one embodiment, a modified GDF-8 propeptide of the inventioncomprises a fusion between a human GDF-8 propeptide (with an inactivatedcleavage site) and an IgG molecule (which IgG acts as the stabilizerprotein or stabilizer portion). As used herein, in addition to referringto a second protein of a fusion protein, a “stabilizer portion” alsoincludes nonproteinaceous modifications such as a carbohydrate moiety,or nonproteinaceous polymer.

The terms “isolated” or “purified” refer to a molecule that issubstantially free of its natural environment. For instance, an isolatedprotein is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which it isderived. The phrase “substantially free of cellular material” refers topreparations where the isolated protein is at least 70% to 80% (w/w)pure, more preferably at least 80-89% (w/w) pure, even more preferably90-95% pure, and most preferably at least 96%, 97%, 98%, 99% or 100%(w/w) pure.

The term “cleavage site” refers to a proteolytic site in a protein orpolypeptide that is acted upon by a proteolytic enzyme resulting in thecleavage of the peptide bond. In one embodiment, a cleavage site of theinvention is “RSRR” as represented by the one-letter amino acid code.

The term “Fc region of an IgG molecule” refers to the Fc domain of animmunoglobulin of the isotype IgG, as is well known to those skilled inthe art. The Fc region of an IgG molecule is that portion of IgGmolecule (IgG1, IgG2, IgG3, and IgG4) that is responsible for increasingthe in vivo serum half-life of the IgG molecule. Preferably, the IgGmolecule is IgG1.

“In vitro half life” refers to the stability of a protein measuredoutside the context of a living organism. Assays to measure in vitrohalf life are well known in the art and include but are not limited toSDS-PAGE, ELISA, cell-based assays, pulse-chase, western blotting,northern blotting, etc. There and other useful assays are well known inthe art.

“In vivo half life” refers to the stability of a protein in an organism.In vivo half life may be measured by a number of methods known in theart including but not limited to in vivo serum half life, circulatoryhalf life, and assays set forth in the examples herein.

“In vivo serum half life” refers to the half-life of a proteincirculating in the blood of an organism. In one embodiment, in vivo halflife is measured as set forth in Example 8. Other methods known in theart may be used to measure in vivo half life.

The term “mammal” includes definitions known in the art and also refersto any animal classified as a mammal, including humans, domestic andfarm animals, and zoo, sports, or pet animals, such as dogs, cats,sheep, pigs, cows, etc. In a preferred embodiment, the mammal is human.

The term “TGF-β superfamily” refers to a family of structurally relatedgrowth factors, all of which possess physiologically importantgrowth-regulatory and morphogenetic properties. This family of relatedgrowth factors is well known in the art (Kingsley et al. (1994) GenesDev. 8:133-46; and Hoodless et al. (1998) Curr. Topics Microbiol.Immunol. 228:235-72). The TGF-β superfamily includes Bone MorphogeneticProtiens (BMPs), Activins, Inhibins, Mullerian Inhibiting Substance,Glial-Derived Neurotrophic Factor, and a still growing number of Growthand Differentiation Factors (GDFs), suck as GDF-8 (myostatin). Many ofthese proteins are related in structure to GDF-8, such as BMP-11 (alsoknown as GDF-11), and/or activity, such as activin.

The term “treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Those in need of treatment mayinclude individuals already having a particular medical disorder as wellas those who may ultimately acquire the disorder (i.e., those needingpreventative measures).

The terms “transformation” and “transfection” refer to a variety ofart-recognized techniques for introducing foreign nucleic acid (e.g.,DNA and RNA) into a host cell, including but not limited to calciumphosphate or calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, and electroporation. SEQ ID NO: NAME TYPE 1human GDF-8 precursor protein protein 2 human GDF-8 precursor proteinDNA 3 human GDF-8 mature protein 4 human GDF-8 mature DNA 5 human GDF-8propetide protein 6 human GDF-8 propeptide DNA 7 human BMP-11 precursorprotein protein 8 human BMP-11 precursor protein DNA 9 human BMP-11mature protein 10 human BMP-11 mature DNA 11 human BMP-11 propeptideprotein 12 human BMP-11 propeptide DNA 13 GDF-8 signal sequence protein14 BMP-11 signal sequence protein 15 IgG-Fc protein 16 IgG-Fc modifiedprotein

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery that GDFpropeptides have a short in vivo half-life thereby reducing theireffectiveness as pharmocologic inhibitors of GDF-8 or BMP-11 activity.Accordingly, in one aspect, the invention features modified andstabilized GDF-8 or BMP-11 propeptide having improved pharmacokineticproperties, specifically an increased circulatory half-life.

The present invention provides novel modified GDF propeptides that forminactive complexes with GDF proteins (for example GDF-8 and BMP-11proteins) in vitro and in vivo. The modified GDF propeptides of theinvention preferably bind to a propeptide binding domain on the matureGDF protein. Accordingly, in certain embodiments of the invention, themodified GDF propeptide comprises a fusion protein between a GDFpropeptide and a stabilizer protein. A stabilizer protein may be anyprotein which enhances the overall stability of the modified GDFpropeptide. As will be recognized by one of ordinary skill in the art,such fusion protein may optionally comprise a linker peptide between thepropeptide portion and the stabilizing portion. As is well known in theart, fusion proteins are prepared such that the second protein is fusedin frame with the first protein such that the resulting translatedprotein comprises both the first and second proteins. For example, inthe present invention, a fusion protein may be prepared such that theGDF propeptide portion is fused to a second protein (e.g. a stabilizerprotein portion.) Such fusion protein is prepared such that theresulting translated protein contains both the propeptide portion andthe stabilizer portion.

In other embodiments, the GDF propeptide comprises a GDF propeptide thatis modified to have greater in vivo circulatory half-life compared tounmodified GDF propeptide. Although the precise mechanism and timing ofmodified GDF propeptide binding is unknown, the modified GDF-8propeptides and modified BMP-11 propeptides of the invention arebelieved to bind to the mature GDF-8 and mature BMP-11. In a preferredembodiment, the present invention provides modified GDF propeptides thatform inactive complexes with GDF-8 or BMP-11 proteins in vivo or invitro. In one embodiment, the GDF propeptides preferably bind to apropeptide binding domain on the mature GDF-8 or mature BMP-11 protein.Such binding may occur, for example, following release of nativepropeptide at the site of GDF-8 and BMP-11 activity, followingdisplacement of the native propeptide by the modified GDF-8 and modifiedBMP-11 propeptide, and/or following cleavage of propeptide from theGDF-8 and BMP-11 precursor protein. Regardless of the mechanism,modified GDF-8 propeptide and/or modified BMP-11 propeptide bindingresults in a reduction in one or more of the biological activitiesassociated with GDF-8, relative to a mature GDF-8 protein that is notbound by the same modified propeptide. In one preferred embodiment, themodified GDF-8 and BMP-11 propeptides reduce GDF-8 and BMP-11 activityassociated with negative regulation of skeletal muscle mass.

In another preferred embodiment, the present invention provides modifiedGDF propeptides that form inactive complexes with BMP-11 proteins invivo or in vitro. In one embodiment, the GDF propeptides preferably bindto a propeptide binding domain on the mature BMP-11 protein. In yetanother embodiment, the modified GDF propeptide is a fusion proteincomprising a GDF propeptide and an Fc region of an IgG molecule (as astabilizing protein). Such GDF inhibitors may comprise a GDF propeptide(for example as set forth in SEQ ID NO:5 or 11) or a fragment or variantof said propeptide which retains one or more biological activities of aGDF propeptide. Such modified GDF propeptides are capable of inhibitingthe activity of GDF proteins. The GDF-8 or BMP-11 propeptides used inthe invention may be synthetically produced, derived from naturallyoccurring (native) GDF-8 or BMP-11 propeptides, or be producedrecombinantly, using any of a variety of reagents, host cells andmethods which are well known in the art of genetic engineering. In oneembodiment, the modified GDF-8 propeptide or modified BMP-11 propeptidecomprises a human GDF-8 or BMP-11 propeptide covalently linked to an IgGmolecule or a fragment thereof. The GDF-8 or BMP-11 propeptide may befused adjacent to the Fc region of the IgG molecule, or attached to theFc region of the IgG molecule via a linker peptide. Use of such linkerpeptides is well known in the protein biochemistry art.

In certain embodiments where the modified GDF propeptides of theinvention comprise fusion proteins, the GDF propeptide portion of saidfusion protein is preferably at least about 75-80% identical to SEQ IDNO: 5 or 11, more preferably at least about 81% to about 85% identicalto SEQ ID NO: 5 or 11, even more preferably at least about 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, or 94% identical to SEQ ID NO: 5 or 11,and even more preferably at least about 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 5 or 11. In a preferred embodiment, the GDF-8 orBMP-11 propeptides comprise a sequence identical to the sequences setforth in SEQ ID NO: 5 or 11. In yet another preferred embodiment, theGDF propeptide portion of a fusion protein of the invention may comprisea fragment of variant of the GDF propeptide set forth in SEQ ID NO: 5 or11, so long as the fragment or variant retains one or more biologicalactivities of a GDF propeptide. In another preferred embodiment, themodified GDF-8 or BMP-11 propeptides comprise a mutant version of SEQ IDNO: 5 or 11, wherein the mutant has been modified to include one or moremutations (including insertion, deletion, or substitution) so long asthe fragment or variant retains one or more biological activities of aGDF propeptide.

Critically, in embodiments of the invention involving modified GDFpropeptides comprising fusion proteins, it is essential that the fusionprotein be produced or designed such that the native proteolyticcleavage site (e.g. RSRR (SEQ ID NO:18)) in the GDF propeptide isdisrupted, destroyed, inactivated or removed in the resulting fusionprotein. As one of skill in the art will recognize, failure to do sowould result in the second protein (e.g., the stabilizer portion) of thefusion protein being cleaved from the first protein (the propeptideportion) of the fusion protein. Accordingly, a critical aspect of theinvention provides for inactivating or eliminating the cleavage sitenative to GDF propeptides when using such propeptide to prepare a fusionprotein which is a modified GDF propeptide of the invention. Methods forinactivating such proteolytic cleavage sites are known in the art andinclude but are not limited to mutation, deletion, or insertion of theamino acid or nucleotide sequence of the proteolytic cleavage site.

In yet other embodiments of the invention, mutations may be made in theGDF sequence to make the sites less susceptible to proteolytic cleavage.For example, in one embodiment, such mutant may contain a point mutationat amino acids 45, 46, 49, 50, 52, 53, 63, 64, 65, 66, 98 or 99 of SEQID NO: 1, or at amino acids 122, 123, 286 or 287 of SEQ ID NO:7. Inanother embodiment, such point mutations can be made in the amino acidsof SEQ ID NO.:11. In a preferred embodiment, the point mutation is apoint mutation at amino acid 99 of SEQ ID NO:1. In a particularlypreferred embodiment, the point mutation is a point mutation of aminoacid 99 of SEQ ID NO.: 1 from Asp to Ala. In another preferredembodiment, such point mutation can be made in the amino acids of SEQ IDNO.: 11. Computer analysis (using a commercially available software,e.g., MacVector, Omega, PCGene, Molecular Simulation, Inc.) can also beused to identify proteolytic cleavage sites. As will be recognized byone of skill in the art any of the described mutations, variants ormodifications may alternatively be made at the nucleic acid level. Suchtechniques are well known in the art. TABLE 2 Exemplary Amino AcidsMutatable to Prevent Cleavage of Propetide: GDF-8 (Amino acid numbersrefer to SEQ ID NO: 1) Arg-45 Gln-46 Lys-49 Ser-50 Arg-52 Ile-53 Lys-63Leu-64 Arg-65 Leu-66 Arg-98 Asp-99 BMP-11 (Amino acids numbers refer toSEQ ID NO: 7) Asp-122 Ala-123 Glu-286 Leu-287

As noted above, the present inventors have made the discovery that theGDF propeptides have a short in vivo half-life. Thus, to bepharmaceutically useful as a therapeutic or prophylactic agent, the GDFpropeptide must be chemically or physically stabilized for increasedlongevity under physiological conditions.

GDF propeptides may be stabilized or modified by any means known in theart, so long as the modified GDF propeptide maintains an ability toinhibit a GDF protein or mature GDF protein. In a preferred embodiment,the modified GDF propeptide maintains its ability to bind to its targetGDF protein or a GDF propeptide binding site. In one preferredembodiment, the modified GDF propeptide comprises a GDF-8 propeptide orBMP-11 propeptide fused adjacent to or via a linker, to an Fc region ofan IgG molecule. The Fc region of the IgG provides a protective orstabilizing effect on the propeptide (and thereby acts as a stabilizingmodification), as reflected in the improved pharmacokinetic properties(i.e., increased serum half-life) of the fusion protein as compared tothe corresponding unmodified GDF-8 propeptide or unmodified BMP-11propeptide.

In a particular embodiment, the modified GDF-8 propeptide comprises ahuman GDF-8 propeptide or a mutant of human GDF-8 propeptide, and theIgG molecule is the Fc region of an IgG1, or an IgG4, or an Fc region ofIgG1 modified for reduced effector function. In one embodiment, the IgGfragment is IgG1 (SEQ ID NO: 15). In another embodiment, the IgGfragment is IgG1 modified for reduced effector function (SEQ ID NO:16).Examples of molecules modified for reduced effector function includemodification of human IgG1-Fc fusion to include alanine (A) at aminoacid positions 14 and 17 as set forth in SEQ ID NO.:16.

Modified GDP-8 or BMP-11 propeptides may be isolated or purifiedaccording to standard protein isolation and purification procedures wellknown in the art.

In another embodiment, the modified GDF propeptide comprises a humanBMP-11 propeptide or a mutant of human BMP-11 propeptide, and an IgGmolecule which is the Fc region of an IgG1, or an IgG4, or an IgG1fragment modified for reduced effector function. In one preferredembodiment, the stabilizer is the Fc region of human IgG1 (SEQ ID NO:15) or the Fc region of human IgG1 modified for reduced effectorfunction (SEQ ID NO: 16). The BMP-11 propeptide may be fused adjacent toor via a linker, to an Fc region of an IgG molecule, as describedherein.

The present invention also provides methods for making modified GDFpropeptides having increased in vivo or in vitro half-life. In onepreferred embodiment, the method comprises preparing a modified GDFpropeptide comprising a GDF-8/IgG1 Fc or BMP-11/IgG1 Fc fusion proteinby preparing a cDNA molecule encoding:

(1) a GDF propeptide (for example human GDF-8 propeptide, SEQ ID NO:5),or human BMP-11 propeptide, SEQ ID NO:11) which is modified toinactivate the proteolytic cleavage site and the Fc region of an IgGmolecule (for example, human IgG1 Fc region, SEQ ID NO:15);

(2) expressing the cDNA in an appropriate prokaryotic or eukaryoticexpression system using appropriate expression plasmids and cells; and

(3) isolating and purifying the expressed fusion protein containing themodified GDF propeptide, wherein the modified GDF propeptide has anincreased in vivo or in vitro half-life as compared to an unmodified GDFpropeptide.

An example of such preferred modified GDF propeptide of the invention isset forth is FIG. 11A.

cDNA expression systems are well known in the molecular biology art asare methods for isolation and purification of the expressed proteins(Examples 2 and 7, herein, provide examples of such embodiments).

In an alternate embodiment, the cDNA constructs used for expression ofsuch fusion proteins may contain nucleotides encoding a linker peptidebetween the nucleotides encoding the GDF propeptide and the IgG Fcregion (or stabilizer portion). The orientation of the linker peptiderelative to the GDF or IgG Fc region is unimportant. The linker peptidemay comprise nucleotides encoding 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30 or more amino acids in length. In a particularembodiment, the linker peptide comprises nucleotides encoding for theamino acid sequence consisting of glycine-serine-glycine-serine (GSGS,SEQ ID NO:17).

In another embodiment, the expression plasmids may optionally containtags allowing for convenient isolation and purification of the expressedproteins. The use of such tagged expression plasmids and the methods forisolating and purifying the tagged protein products are well known inthe art.

The GDF propeptides of the invention specifically include fusionproteins in which the GDF propeptide portion of the molecule isidentical or greater than 60% homologous to GDF corresponding sequencesfrom a particular species, while the stabilizer portion, such as the Fcregion of the IgG molecule may be identical or greater than 60%homologous to IgG corresponding sequences from a different species. Forexample, the human GDF-8 propeptide or fragment thereof may be fused toan Fc region of a mouse IgG molecule, or vice versa, so long as theresulting fusion protein exhibits the desired biological activity,namely, the inhibition of GDF biological activity, as embodied inExamples 3-6. In preferred embodiments of the invention, fusion proteinsof the invention are chimeras of human proteins. As will be understoodin the art, human protein chimeric or fusion proteins will be preferredin the treatment of human subjects.

As an alternative or in addition to the above-described Fc-fusionproteins, the GDF propeptides may be stabilized by a variety of othermethods and resource materials which are well known and readilyavailable in the protein art. Such methods and materials include, forexample, glycosylation, or linkage to albumin or a nonproteineouspolymer, as described in detail below. The modified GDF propeptides maybe isolated and purified using standard protein isolation andpurification techniques well known in the protein art.

GDF propeptides or modified GDF propeptides can be produced by a varietyof art-known techniques. For example, such propeptides can besynthesized (e.g., chemically or recombinantly), isolated and purified,and tested for their ability to form complexes with mature GDF-8 ormature BMP-11 protein using the methods described herein or methodsknown in the art. Propeptides can be synthesized using standard proteinchemistry techniques such as those described in Bodansky, M. Principlesof Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant G.A.(ed.), Synthetic Peptides: A User's Guide, W. H. Freeman and Company,New York (1992), the contents of which are incorporated herein byreference. In addition, automated peptide synthesizers are commerciallyavailable (e.g., Advanced ChemTech Model 396; Milligen/Biosearch 9600).

Alternatively, the modified or unmodified GDF propeptides or fragmentsthereof may be recombinately produced using various expression systems(e.g., E. coli, Chinese Hamster Ovary cells, COS cells, baculovirus) asis well known in the art. For example, the expressed propeptide of GDF-8or BMP-11 may be purified by, for example, using the method described inBottinger et al. (1996) PNAS 93:5877-5882 and Gentry and Nash (1990)Biochemistty 29:6851-6857, the contents of which are incorporated hereinby reference, or any other art recognized method for purifying peptides.Alternatively, the modified or unmodified GDF propeptide or fragmentthereof may be tagged with, for example, FLAG or 6-His for subsequentpurification using protein art-established techniques.

The modified or unmodified GDF-8 or BMP-11 propeptides may further beproduced by digestion of naturally occurring or recombinantly producedGDF-8 or BMP-11 using, for example, a protease, e.g., trypsin,thermolysin, chymotrypsin, pepsin, or paired basic amino acid convertingenzyme (PACE). Computer analysis (using a commercially availablesoftware, e.g., MacVector, Omega, PCGene, Molecular Simulation, Inc.)can be used to identify proteolytic cleavage sites. Alternatively, suchGDF propeptides may be produced from naturally occurring orrecombinantly produced GDF-8 or BMP-11 such as standard techniques knownin the art, such as by chemical cleavage (e.g., cyanogen bromide,hydroxylamine).

The proteolytic or synthetic GDF-8 and BMP-11 propeptide portions of themodified GDF propeptide may comprise as many amino acid residues as arenecessary to bind to the target GDF protein, thereby inhibiting,partially or completely, GDF-8 or BMP-11 activity. Examples 4-6, herein,illustrate embodiments of binding and inhibition assays. In particular,functional fragments of GDF-8 and/or BMP-11 propeptide sequences thatmaintain the ability to modulate or inhibit GDF-8, are included withinthe scope of the invention. The GDF-8 propeptide portions preferablycomprise at least 5, 10, 20, 30, 40, 50, 60, 70, 80, or 90 amino acids;more preferably at least 100, 110, 120, 130, 140, 150, 160, 170, 180, or190 amino acids; and most preferably at least 200, 210, 220, 230, or 240or more amino acids in length. In a preferred embodiment, the GDF-8propeptide portion of the modified GDF-8 propeptide is 243 amino acidsin length, i.e., corresponding to SEQ ID NO:5; and the BMP-11 propeptideportion of the BMP-11 propeptide is 274 amino acids in length, i.e.,corresponding to SEQ ID NO:11. The signal sequence for human GDF-8 isset forth in SEQ ID NO:13, and includes the first 23 amino acids of SEQID NO:1. In one embodiment, the sequence of BMP-11 propeptide beginswith the amino acid sequence AEGPAAA (SEQ ID NO:19).

The signal sequence for human BMP-11 is set forth in SEQ ID NO.: 14 andincludes the first 24 amino acids of SEQ ID NO.: 7. The identificationof a partial GDF-8 or BMP-11 propeptide sequence as a functionalfragment of GDF-8 or BMP-11 propeptide may readily be determined, forexample, using the assays described in Examples 4-6 herein.

In addition to truncated propeptide sequences, the propeptide variantsherein specifically include GDF propeptides having point mutations orother modifications (including insertion, deletion, and substitution);so long as such variants contain one or more desired biological orinhibitory activities of a GDF propeptide. Such activity can bemeasured, for example, as described in Examples 4-6. Such modificationsmay be introduced into the molecule to enhance the activity, circulatoryor storage half-life, or production of the GDF-8 or BMP-11 propeptide.For example, point mutations may be introduced into one or moreproteolytic cleavage sites to prevent or inhibit proteolytic degradationof the modified GDF-8 propeptide in vivo. Computer analysis (using acommercially available software, e.g., MacVector, Omega, PCGene,Molecular Simulation, Inc.) can be used to identify proteolytic cleavagesites.

Accordingly, methods of making GDF propeptides of the inventionspecifically include, in addition to the wild-type cDNA codingsequences, cDNA coding sequences that encode the wild-type GDFpropeptides but which differ in cDNA sequence from the wild-type GDFcDNA sequence due to the degeneracies of the genetic code or allelicvariations (naturally-occurring base changes in the species populationwhich may or may not result in an amino acid change). The invention alsocontemplates DNA sequences that hybridize under stringent hybridizationconditions (in one embodiment as described in T. Maniatis et al. (1982)Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory,pp. 387-389) to a nucleic acid encoding a GDF propeptide or a nucleicacid encoding a protein or polypeptide having the ability to bind to aparticular GDF protein, as embodied in Examples 4-6. Variations in thecDNA sequences caused by point mutations or by induced modifications(e.g., insertion, deletion, and substitution) to enhance the activity,half-life or production of the GDF-8 or BMP-11 propeptides encodedthereby are also useful for the present invention. Computer programsuseful in the determination of DNA sequence homology are known in theart and are described herein.

The ability of a modified GDF propeptide to form a noncovalent complexwith a GDF protein can be evaluated by a variety of methods known in theart, including size exclusion chromatography and/or cross linkinganalyses, as described in Example 2 herein. As shown in Example 2, theGDF-8 propeptide forms a noncovalent association with mature GDF-8protein. The mature GDF-8/GDF-8 propeptide complex has an apparentmolecular weight of approximately 75 kDa.

As stated above, in addition to the Fc fusion method, GDF propeptidesmay be stabilized by a number of other techniques. In one embodiment, astabilizer portion is covalently linked to a GDF propeptide portion tocreate a fusion protein. For example, the GDF-8 propeptide may be linkedto one or more of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337, incorporated by reference herein. Incertain embodiments, GDF propeptides are chemically modified by covalentconjugation to a polymer to increase their circulating half-life.Preferred polymers, and methods to attach them to peptides, are alsodescribed in U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285; and4,609,546, incorporated by reference herein.

In another embodiment, the GDF-8 propeptide may be modified to have analtered glycosylation pattern (i.e., altered from the wild-typeglycosylation pattern). As used herein, “altered” means having one ormore carbohydrate moieties added or deleted, to/from the wild-type GDFpropeptide. Glycosylation of proteins and polypeptides is typicallyeither N-linked or O-linked. N-linked refers to the attachment of thecarbohydrate moiety to the side chain of an asparagine residue. Thetripeptide sequences asparagine-X-serine and asparagine-X-threonine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. Thus, the presence of either of these tripeptide sequencesin a polypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition or deletion of glycosylation sites to the GDF propeptide isconveniently accomplished by altering the amino acid sequence such thatit contains or omits one or more of the above-described tripeptidesequences (for N-linked glycosylation sites). The alteration may also bemade by the addition of, or substitution by, one or more serine orthreonine residues to the sequence of the wild-type GDF propeptide (forO-linked glycosylation sites). For ease, the GDF propeptide amino acidsequence is preferably altered through changes at the DNA level, whichtechniques are well known in the art.

Another means of increasing the number of carbohydrate moieties on theGDF propeptide is by chemical or enzymatic coupling of glycosides to theGDF propeptide. These procedures are advantageous in that they do notrequire production of the GDF propeptide in a host cell that hasglycosylation capabilities for N- or O-linked glycosylation. Dependingon the coupling mode used, the sugar(s) may be attached to (a) arginineand histidine; (b) free carboxyl groups; (c) free sulfhydryl-groups suchas those of cysteine; (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline; (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan; or (f) the amide group ofglutamine. These methods are described in WO 87/05330 published 11 Sep.1987, and in Aplin and Wriston (1981) CRC Crit. Rev. Biochem., pp.259-306, incorporated by reference herein.

Removal of any carbohydrate moieties present on the GDF propeptide maybe accomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the GDF propeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving the aminoacid sequence intact.

Chemical deglycosylation is described by Hakimuddin et al. (1987) Arch.Biochem. Biophys. 259:52 and by Edge et al. (1981) Anal. Biochem.118:131. Enzymatic cleavage of carbohydrate moieties on GDF propeptidescan be achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al. (1987) Meth. Enzymol. 138:350.

When the modified GDF propeptide is a GDF propeptide-Fc fusion protein,as described above, the Fc region of the IgG molecule may also bemodified to have an altered glycosylation pattern.

In another embodiment, a GDF propeptide is linked to the protein albuminor a derivative of albumin. Methods for linking proteins andpolypeptides to protein albumin or albumin derivatives are well known inthe art. See e.g., U.S. Pat. No. 5,116,944 (Sivam et al.), incorporatedby reference herein.

It is understood by one of ordinary skill in the art that certain aminoacids may be substituted for other amino acids in a protein structurewithout adversely affecting the activity of the protein. It is thuscontemplated by the inventors that various changes may be made in theamino acid sequences of the modified or unmodified GDF propeptides, orDNA sequences encoding such propeptides, without appreciable loss oftheir biological utility or activity. In one embodiment, such activityis measured in Examples 4-6. Such changes may include, but are notlimited to, deletions, insertions, truncations, substitutions, fusions,and the like. For example, alterations of amino acid sequences atproteolytic cleavage sites within the modified GDF propeptide areexplicitly encompassed within the present invention.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biological function on a protein is generallyunderstood in the art (Kyte and Doolittle (1982) J. Mol. Biol.157:105-132). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis ofits hydrophobicity and charge characteristics (Kyte and Doolittle, 1982,supra); these are isoleucine (+4.5), valine (+4.2), leucine (+3.8),phenylalanine (+2.8), cysteine/cystine (+2.5), methionine (+1.9),alanine (+1.8), glycine (−0.4), threonine (−0.7), serine (−0.8),tryptophan (−0.9), tyrosine (−1.3), proline (−1.6), histidine (−3.2),glutamate (−3.5), glutamine (−3.5), aspartate (−3.5), asparagine (−3.5),lysine (−3.9), and arginine (−4.5).

In making such changes, the substitution of amino acids whosehydropathic indices are within ±2 is preferred, those which are within±1 are particularly preferred, and those within ±0.5 are even moreparticularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. Forexample, U.S. Pat. No. 4,554,101 states that the greatest local averagehydrophilicity of a protein, as govern by the hydrophilicity of itsadjacent amino acids, correlates with a biological property of theprotein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0),lysine (+3.0), aspartate (+3.0±1), glutamate (+3.0±1) serine (+0.3),asparagine (+0.2), glutamine (+0.2), glycine (0), threonine (−0.4),proline (−0.5±1), alanine (−0.5), histidine (−0.5), cysteine (−1.0),methionine (−1.3), valine (−1.5), leucine (−1.8), isoleucine (−1.8),tyrosine (−2.3), phenylalanine (−2.5), and tryptophan (−3.4).

In making such changes, the substitution of amino acids whosehydrophilicity values are within ±2 is preferred, those which are within±1 are particularly preferred, and those within ±0.5 are even moreparticularly preferred.

The modifications may be conservative such that the structure orbiological function of the protein is not affected by the change. Suchconservative amino acid modifications are based on the relativesimilarity of the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. Exemplaryconservative substitutions which take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine, andisoleucine. The amino acid sequence of the modified GDF propeptides maybe modified to have any number of conservative changes, so long as thebinding of the modified GDF propeptide to its target GDF protein is notadversely affected.

Relative sequence similarity or identity (also known in the protein andmolecular biology arts as sequence homology) is preferably determinedusing the “Best, Fit” or “Gap” programs of the Sequence AnalysisSoftware Package™ (Version 10; Genetics Computer Group, Inc., Universityof Wisconsin Biotechnology Center, Madison, Wis.). “Gap” utilizes thealgorithm of Needleman and Wunsch (Needleman and Wunsch, 1970) to findthe alignment of two sequences that maximizes the number of matches andminimizes the number of gaps. “BestFit” performs an optimal alignment ofthe best segment of similarity between two sequences. Optimal alignmentsare found by inserting gaps to maximize the number of matches using thelocal homology algorithm of Smith and Waterman (Smith and Waterman,1981; Smith, et al., 1983).

The Sequence Analysis Software Package described above contains a numberof other useful sequence analysis tools for identifying homologues ofthe presently disclosed nucleotide and amino acid sequences. Forexample, the “BLAST” program (Altschul, et al., 1990) searches forsequences similar to a query sequence (either peptide or nucleic acid)in a specified database (e.g., sequence databases maintained at theNational Center for Biotechnology Information (NCBI) in Bethesda, Md.,USA); “FastA” (Lipman and Pearson, 1985; see also Pearson and Lipman,1988; Pearson, et al., 1990) performs a Pearson and Lipman search forsimilarity between a query sequence and a group of sequences of the sametype (nucleic acid or protein); “TfastA” performs a Pearson and Lipmansearch for similarity between a protein query sequence and any group ofnucleotide sequences (it translates the nucleotide sequences in all sixreading frames before performing the comparison); “FastX” performs aPearson and Lipman search for similarity between a nucleotide querysequence and a group of protein sequences, taking frameshifts intoaccount. “TfastX” performs a Pearson and Lipman search for similaritybetween a protein query sequence and any group of nucleotide sequences,taking frameshifts into account (it translates both strands of thenucleic sequence before performing the comparison).

One of skill in the art will recognize that the modified or unmodifiedGDF propeptides may contain any number of substitutions to their aminoacid sequences without altering their biological properties. In apreferred embodiment, such changes are conservative amino acidsubstitutions, that are well known in the art. Exemplary conservativesubstitutions which take various of the foregoing characteristics intoconsideration are well known to those of skill in the proteinbiochemistry art and include but are not limited to: arginine andlysine; glutamate and aspartate; serine and threonine; glutamine andasparagine; and valine, leucine, and isoleucine.

In certain embodiments of the invention, the in vivo stability of aprotein can be measured in a number of ways. In one embodiment, serum ortissue samples are harvested at a variety of time points followingdelivery of the protein either intravenously or intraperitoneally or theprotein is radiolabeled, i.e. with iodine-125, using methods well knownto those in the art. The amount of radioactivity present in each serumor tissue sample can be determined using a gamma-counter. In anotherembodiment, the integrity/stability of the protein in the serum can beassessed by SDS-PAGE followed by either autoradiography or Western blotanalysis. In another embodiment, the biological activity of the proteincan be measured using any one of a number of functional assays,including an ELISA or cell-based assay known in the art.

Methods of Treating Disease

The GDF propeptides of the present invention are useful to prevent ortreat various medical disorders in humans or animals. The GDFpropeptides are preferably used to inhibit or reduce one or moreactivities associated with a GDF protein. In one highly preferredembodiment, the modified GDF propeptide inhibits or reduces one or moreof the activities of mature GDF-8 relative to a mature GDF-8 proteinthat is not bound by the same propeptide. In a preferred embodiment, theactivity of the mature GDF-8 protein, when bound by one or more of themodified GDF propeptides, is inhibited at least 50%, preferably at least60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, or 88%, morepreferably at least 90, 91, 92, 93, or 94%, and even more preferably atleast 95% to 100% relative to a mature GDF-8 protein that is not boundby one or more of the modified GDF propeptides.

The medical disorder being treated or prevented by the modified GDFpropeptides is preferably a muscle or neuromuscular disorder (such asamyotrophic lateral sclerosis, muscular dystrophy, muscle atrophy,congestive obstructive pulmonary disease, muscle wasting syndrome,sarcopenia, or cachexia), a metabolic disease (such as such as type 2diabetes, noninsulin-dependent diabetes mellitus, hyperglycemia, orobesity), an adipose tissue disorder (such as obesity), or bonedegenerative disease (such as osteoporosis). The medical condition ismost preferably a muscle or neuromuscular disorder, such as amyotrophiclateral sclerosis, muscular dystrophy, muscle atrophy, congestiveobstructive pulmonary disease, muscle wasting syndrome, sarcopenia, orcachexia. In another preferred embodiment, the medical condition is ametabolic disease or disorder, such as that resulting from dysfunctionalglucose metabolism and/or insulin resistance, such as type 2 diabetes ornoninsulin-dependent diabetes mellitus, or metabolic disorders such ashyperglycemia or obesity. The GDF propeptides are preferably used toprevent or treat such medical disorders in mammals, most preferably inhumans.

The GDF propeptides or propeptides compositions of the present inventionare administered in therapeutically effective amounts. As used herein,an “effective amount” of the modified GDF propeptide is a dosage whichis sufficient to reduce the activity of GDF proteins to achieve adesired biological outcome (e.g., increasing skeletal muscle mass). Inone embodiment, the desired biological outcome may be any therapeuticbenefit including an increase in muscle mass, an increase in musclestrength, improved metabolism, decreased adiposity, or improved glucosehomeostasis. Such improvements may be measured by a variety of methodsincluding those that measure lean and fat body mass (such as duel x-rayscanning analysis), muscle strength, serum lipids, serum leptin, serumglucose, glycated hemoglobin, glucose tolerance, and improvement in thesecondary complication of diabetes. Generally, a therapeuticallyeffective amount may vary with the subject's age, weight, physicalcondition, and sex, as well as the severity of the medical condition inthe subject. The dosage may be determined by a physician and adjusted,as necessary, to suit observed effects of the treatment. Generally, thecompositions are administered so that GDF propeptides are given at adose between 50 μg/kg and 20 mg/kg. Preferably, the GDF propeptides aregiven as a bolus dose, to maximize the circulating levels of GDFpropeptides for the greatest length of time after the dose. Continuousinfusion may also be used after the bolus dose.

The present invention also provides methods for preventing or treatingmetabolic diseases or disorders resulting from abnormalglucosehomeostasis. Normal glucose homeostasis requires, inter alia, thefinely tuned orchestration of insulin secretion by pancreatic beta cellsin response to subtle changes in blood glucose levels. One of thefundamental actions of insulin is to stimulate uptake of glucose fromthe blood into tissues, especially muscle and fat.

Accordingly, in one embodiment, the present invention provides a methodfor treating diabetes mellitus and related disorders, such as obesity orhyperglycemia, by administering to a subject a modified GDF propeptideor modified GDF propeptide composition in an amount sufficient toameliorate the symptoms of the disease. Type 2 or noninsulin-dependentdiabetes mellitus (NIDDM), in particular, is characterized by a triad of(1) resistance to insulin action on glucose uptake in peripheraltissues, especially skeletal muscle and adipocytes, (2) impaired insulinaction to inhibit hepatic glucose production, and (3) dysregulatedinsulin secretion (DeFronzo, (1997) Diabetes Rev.5:177-269). Therefore,subjects suffering from type 2 diabetes can be treated according to thepresent invention by administration of a modified GDF propeptide, whichincreases sensitivity to insulin and glucose uptake by cells.

Similarly, other diseases and metabolic disorders characterized byinsulin dysfunction (e.g., resistance, inactivity, or deficiency) and/orinsufficient glucose transport into cells also can be treated accordingto the present invention by administration of a modified GDF propeptide,which increases sensitivity to insulin and glucose uptake by cells.

The modified GDF propeptide or modified GDF propeptide compositions ofthe present invention are administered in therapeutically effectiveamounts. When used for the treatment of diabetes and related disorders,an “effective amount” of the modified GDF propeptide is a dosage whichis sufficient to reduce the activity of GDF proteins to achieve one ormore desired therapeutic results, such as, for example, an increase ininsulin sensitivity or glucose uptake by cells, a decrease in fat bodymass or a desired change in serum lipids, serum leptin, serum glucose,glycated hemoglobin, glucose tolerance, or improvement in the secondarycomplication of diabetes. Generally, a therapeutically effective amountmay vary with the subject's age, condition, and sex, as well as theseverity of the insulin dysfunction in the subject. The dosage may bedetermined by a physician and adjusted, as necessary, to suit observedeffects of the treatment. Generally, the compositions are administeredso that GDF propeptides are given at a dose between 50 ug/kg and 20mg/kg. Preferably, the GDF propeptides are given as a bolus dose, tomaximize the circulating levels of GDF propeptides for the greatestlength of time after the dose. Continuous infusion may also be usedafter the bolus dose.

The present invention also provides gene therapy for the in vivoproduction of GDF propeptides. Such therapy would achieve itstherapeutic effect by introduction of the GDF propeptide polynucleotidesequences into cells or tissues having the disorders as listed above.Delivery of GDF propeptide polynucleotide sequences can be achievedusing a recombinant expression vector such as a chimeric virus or acolloidal dispersion system. Especially preferred for therapeuticdelivery of GDF propeptide polynucleotide sequences is the use oftargeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated By inserting a GDF propeptide polynucleotidesequence of interest into the viral vector, along with another genewhich encodes the ligand for a receptor on a specific target cell, forexample, the vector is now target specific. Retroviral vectors can bemade target specific by attaching, for example, a sugar, a glycolipid,or a protein. Preferred targeting is accomplished by using an antibody.Those of skill in the art will recognize that specific polynucleotidesequences can be inserted into the retroviral genome or attached to aviral envelope to allow target specific delivery of the retroviralvector containing the GDF propeptide polynucleotide. In one preferredembodiment, the vector is targeted to muscle cells or muscle tissue.

Since recombinant retroviruses are defective, they require helper celllines that contain plasmids encoding all of the structural genes of theretrovirus under the control of regulatory sequences within the LTR.These plasmids are missing a nucleotide sequence which enables thepackaging mechanism to recognize an RNA transcript for encapsidation.Helper cell lines which have deletions of the packaging signal include,but are not limited to .PSI.2, PA317 and PA12, for example. These celllines produce empty virions, since no genome is packaged. If aretroviral vector is introduced into such cells in which the packagingsignal is intact, but the structural genes are replaced by other genesof interest, the vector can be packaged and vector virion produced.

Alternatively, other tissue culture cells can be directly transfectedwith plasmids encoding the retroviral structural genes gag, pol and env,by conventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for GDF propeptide polynucleotide is acolloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. RNA, DNA andintact virions can be encapsulated within the aqueous interior and bedelivered to cells in a biologically active form (See e.g., Fraley, etal., Trends Biochem. Sci., 6:77, 1981). Methods for efficient genetransfer using a lipsosome vehicle, are known in the art, see e.g.,Mannino, et al., Biotechniques, 6:682, 1988. The composition of theliposome is usually a combination of phospholipids, usually incombination with steroids, especially cholesterol. Other phospholipidsor other lipids may also be used. The physical characteristics ofliposomes depend on pH, ionic strength, and the presence of divalentcations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Illustrative phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine anddistearoylphosphatidylcholine.

The targeting of liposomes is also possible based on, for example,organ-specificity, cell-specificity, and organelle-specificity and isknown in the art.

The methods of treating or preventing the above medical conditions withthe modified GDF propeptides can also be used to inhibit other proteinsin the TGF-β1 superfamily. Many of these proteins are related instructure to GDF-8, such as BMP-11. Accordingly, in another embodiment,the invention provides methods of treating the aforementioned disordersby administering to a subject a modified GDF propeptide capable ofinhibiting a non-GDF-8 protein, either alone or in combination with amodified GDF propeptide against GDF-8.

Modified GDF Propeptide Compositions

The present invention provides compositions comprising the modified GDFpropeptides. Such compositions may be suitable for pharmaceutical useand administration to patients. The compositions typically comprise oneor more modified GDF propeptides of the present invention and apharmaceutically acceptable excipient. As used herein, the phrase“pharmaceutically acceptable excipient” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, that arecompatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Exemplary pharmaceutically acceptable excipients can be found in “TheHandbook of Pharmaceutical Excipients” 3^(rd) Ed. 2000, Am.Pharmaceutical Press, A.E. Kibbe, ed. The compositions may also containother active compounds providing supplemental, additional, or enhancedtherapeutic functions. The pharmaceutical compositions may also beincluded in a container, pack, or dispenser together with instructionsfor administration.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Methods toaccomplish the administration are known to those of ordinary skill inthe art. It may also be possible to obtain compositions which may betopically or orally administered, or which may be capable oftransmission across mucous membranes. The administration may, forexample, be intravenous (I.V.), intraperitoneal (I.P.), intramuscular(I.M.), intracavity, subcutaneous (S.C.) or transdermal. Preferredembodiments include I.V., I.P., I.M. and S.C. injection. In onepreferred embodiment, the pharmaceutical compositions of the inventionare administered intravenously.

Solutions or suspensions used for intradermal or subcutaneousapplication typically include one or more of the following components: asterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates; and agents for the adjustment oftonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Suchpreparations may be enclosed in ampoules, disposable syringes ormultiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injection include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, Cremophor EL™ (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, thecomposition must be sterile and should be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prevention of theaction of microorganisms can be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as manitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Oral compositions generally include an inert diluent or an ediblecarrier. For example, they can be enclosed in gelatin capsules orcompressed into tablets. For the purpose of oral therapeuticadministration, the GDF propeptides can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Pharmaceuticallycompatible binding agents, and/or adjuvant materials can be included aspart of the composition. The tablets, pills, capsules, troches, and thelike can contain any of the following ingredients, or compounds of asimilar nature; a binder such as microcrystalline cellulose, gumtragacanth or gelatin; an excipient such as starch or lactose, adisintegrating agent such as alginic acid, Primogel, or corn starch; alubricant such as magnesium stearate or Sterotes; a glidant such ascolloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,or orange flavoring.

For administration by inhalation, the GDF propeptides may be deliveredin the form of an aerosol spray from pressured container or dispenserwhich contains a suitable propellant, e.g., a gas such as carbondioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The GDF propeptides may also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

In one embodiment, the modified GDF propeptides are prepared withcarriers that will protect the compound against rapid elimination fromthe body; such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensionscontaining the modified GDF propeptides can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.GDF propeptides which exhibit large therapeutic indices are preferred.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any modified GDFpropeptide used in the present invention, the therapeutically effectivedose can be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test modified GDF propeptide which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Levels in plasmamay be measured, for example, by high performance liquid chromatography.The effects of any particular dosage can be monitored by a suitablebioassay. Examples of suitable bioassays include DNA replication assays,transcription-based assays, GDF protein/receptor binding assays,creatine kinase assays, assays based on the differentiation ofpre-adipocytes, assays based on glucose uptake in adipocytes, andimmunological assays.

The following examples provide preferred embodiments of the invention.One of ordinary skill in the art will recognize the numerousmodifications and variations that may be performed without altering thespirit or scope of the present invention. Such modifications andvariations are believed to be encompassed within the scope of theinvention. The examples do not in any way limit the invention.

The entire contents of all references, patents and published patentapplications cited throughout this application are herein incorporatedby reference.

EXAMPLES Example 1 Purification of GDF-8

Conditioned media from a selected cell line expressing recombinant humanGDF-8 protein (mature GDF-8+GDF-8 propeptide) was acidified to pH 6.5and applied to a 80×50 mm POROS HQ anion exchange column in tandem to a80×50 mm POROS SP cation exchange column (Perseptive Biosystems). Theflow through was adjusted to pH 5.0 and applied to a 75×20 mm POROS SPcation exchange column (Perseptive Biosystems) and eluted with a NaClgradient. Fractions containing the GDF-8, as indicated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), were pooled,acidified with trifluoroacetic acid (TFA) to pH 2-3, then brought up to200 ml with 0.1% TFA to lower the viscosity. The pool was then appliedto a 250×21.2 mm C₅ column (Phenomenex) preceded by a 60×21.2 mm guardcolumn (Phenomenex) and eluted with a TFA/CH₃CN gradient, to separatemature GDF-8 from GDF-8 propeptide. Pooled fractions containing matureGDF-8 were concentrated by lyophilization to remove the acetonitrile and20 ml of 0.1% TFA was added. The sample was then applied to a 250×10 mmC₅ column (Phenomenex) heated to 60° C. to aid in separation. This wasrepeated until further separation could no longer be achieved. Fractionscontaining mature GDF-8 were then pooled and brought up to 40%acetonitrile and applied to a 600×21.2 BioSep S-3000 size exclusioncolumn (Phenomenex) preceded by a 60×21.2 guard column. Fractionscontaining purified mature GDF-8 were pooled and concentrated for use insubsequent experiments. Typical yields of the mature GDF-8 dimer was0.33 mg of protein per liter of conditioned media.

C₅ column fractions containing GDF-8 propeptide were pooled, theacetonitrile was removed by evaporation, 20 ml of 0.1% TFA was added,and the sample was then injected onto the 250×10 mm C₅ column at 60° C.This was repeated until further separation could no longer be achieved.Fractions containing the GDF-8 propeptide were then pooled and broughtup to 40% acetonitrile and applied to a 600×21.2 BioSep S-3000 sizeexclusion column (Phenomenex) preceded by a 60×21.2 guard column.Fractions containing the purified GDF-8 propeptide were pooled andconcentrated for use in subsequent experiments. A typical yield of theGDF-8 propeptide was 0.24 mg protein per liter of conditioned media.

On SDS-PAGE, purified mature GDF-8 migrated as a broad band at 25 kDaunder nonreducing conditions and 13 kDa under reducing conditions. Asimilar SDS-PAGE profile has been reported for murine GDF-8 (McPherronet al. (1997) Nature 387:83-90), and reflects the dimeric nature of themature protein.

The apparent molecular weight of purified GDF-8 propeptide was 38 kDaunder both reducing and nonreducing conditions. This indicates that theGDF-8 propeptide is monomeric. The difference between the apparentmolecular weight and the predicted molecular weight of GDF-8 propeptide,˜26 kDa, may reflect the addition of carbohydrate, since its amino acidsequence contains a potential N-linked glycosylation site (McPherron etal. (1997) Proc. Natl. Acad. Sci. USA 94:12457-12461). Thus, the aboveprocess led to the production of purified and active mature GDF-8 dimerand GDF-8 propeptide.

Example 2 Characteristics of Purified Recombinant Human GDF-8

50 ug each of purified mature GDF-8 and purified GDF-8 propeptide weremixed and dialyzed into 50 mM sodium phosphate, pH 7.0, andchromatographed on a 300×7.8 mm BioSep S-3000 size exclusion column(Phenomenex). Molecular weight of the mature GDF-8/propeptide complexwas determined from elution time, using molecular weight standards(Bio-Rad Laboratories, Hercules, Calif.) chromatographed on the samecolumn.

When purified GDF-8 propeptide was incubated with purified mature GDF-8at neutral pH, the two proteins appeared to complex, as indicated by thesize exclusion profile. The primary protein peak eluted at 12.7 minuteshad an estimated molecular weight of 78 kDa from molecular weightstandards (Bio-Rad Laboratories, Hercules, Calif.) chromatographed onthe same column. The size of the complex is most consistent with onedimer of the mature GDF-8 associating with two monomers of propeptide,thus forming a tetrameric complex.

To confirm this observation, a preparation containing both mature GDF-8and GDF-8 propeptide was incubated with or without 100 mM 1-Ethyl3-[3-dimethylaminopropyl]carbodiamide hydrochloride (EDC, Pierce) for 1hour at room temperature, acidified with HCl to pH 2-3, and concentratedwith a Micron-10 concentrator (Amicon) for SDS-PAGE, using a tricinebuffered 10% acrylamide gel. Proteins were visualized by Coomassie bluestaining. A band corresponding to a molecular weight of about 75 kD wasobserved only in the sample EDC was added and not in the control lane,confirming the presence of the GDF-8 mature/GDF-8 propeptide complex.This valides the assay as useful for measuring the activity andconcentration of purified mature GDF-8 dimer.

Example 3 In Vitro Biological Activity of Purified GDF-8

To demonstrate the activity of GDF-8, a reporter gene assay (RGA) wasdeveloped using a reporter vector pGL3(CAGA)₁₂ sequence coupled toluciferase. The —CAGA sequence was previously reported to be aTGF-β-responsive sequence within the promoter of the TGF-β-induced gene,PAI-1 (Dennler et al. (1998) EMBO J. 17:3091-3100).

The reporter vector containing 12 CAGA boxes was made using the basicreporter plasmid, pGL3 (Promega Corporation, Madison, Wis., USA, Cat.No. E1751). The TATA box and transcription initiation site from theadenovirus major late promoter (−35/+10) was inserted between the BgIIIand HindIII sites. Oligonucleotides containing twelve repeats of theCAGA boxes AGCCAGACA were annealed and cloned into the XhoI site. Thehuman rhabdomyosarcoma cell line, A204 (ATCC HTB-82), was transientlytransfected with pGL3(CAGA)₁₂ using FuGENE 6 transfection reagent (RocheDiagnostics, Indianapolis, USA, Cat. No. 1814443). Followingtransfection, cells were cultured on 48-well plates in McCoy's 5A medium(Life Technologies, Rockville, Md., USA, Cat. No. 21500-079)supplemented with 2 mM glutamine, 100 U/ml streptomycin, 100 μg/mlpenicillin and 10% fetal calf serum for 16 h. Cells were then treatedwith GDF-8, BMP-11 or activin in McCoy's 5A media with glutamine,streptomycin, penicillin and 1 mg/ml bovine serum albumin for 6 h at 37°C. Luciferase was quantified in the treated cells using the LuciferaseAssay System (Promega Corporation, Madison, Wis., USA, Cat. No. E1483).The assay results are illustrated in FIG. 1. For GDF-8, results areexpressed as mean±S.E. for three separate experiments. For BMP-11 andactivin, results are representative of two separate experiments.

The results show that GDF-8 maximally activated the reporter construct10-fold, with an ED₅₀ of 10 ng/ml GDF-8, indicating that the purifiedrecombinant GDF-8 was biologically active. BMP-11, which is 90%identical to GDF-8 at the amino acid level (Gamer et al. (1999) Dev.Biol. 208:222-232 and Nakashima et al. (1999) Mech. Dev. 80:185-189),and activin elicited a similar biological response, indicating that thereporter gene assay is a suitable assay to detect the in vitrobiological activity of GDF-8, BMP-11 or activin.

Example 4 Binding Properties of Purified GDF-8

The GDF-8 latent complex was biotinylated at a ratio of 20 moles ofEZ-link Sulfo-NHS-Biotin (Pierce, Rockford, Ill., USA, Cat. NO. 21217)to 1 mole of the GDF-8 complex for 2 hours on ice, inactivated with 0.5%TFA, and subjected to chromatography on a C4 Jupiter 250×4.6 mm column(Phenomenex) to separate mature GDF-8 from GDF-8 propeptide.Biotinylated mature GDF-8 fractions eluted with a TFA/CH₃CN gradientwere pooled, concentrated and quantitated by MicroBCA protein AssayReagent Kit (Pierce, Rockford, Ill., USA, Cat. No. 23235).

Recombinant ActRIIB-Fc chimera (R&D Systems, Minneapolis, Minn., USACat. No. 339-RB/CF) was coated on 96-well flat-bottom assay plates(Costar, N.Y., Cat. No. 3590) at 1 μg/ml in 0.2 M sodium carbonatebuffer overnight at 4° C. Plates were then blocked with 1 mg/ml bovineserum albumin and washed following standard ELISA protocol. 100 μl ofbiotinylated GDF-8 aliqouts at various concentrations were added to theblocked ELISA plate, incubated for 1 hr, washed, and the amount of boundGDF-8 was detected by Streptavidin-Horseradish peroxidase (SA-HRP, BDPharMingen, San Diego, Calif., USA, Cat. No. 13047E) followed by theaddition of TMB (KPL, Gaithersburg, Md., USA, Cat. No. 50-76-04).Colorimetric measurements were done at 450 nM in a Molecular Devicesmicroplate reader.

As shown in FIG. 2, biotinylated GDF-8 bound to ActRIIB, the putativeGDF-8 type 11 receptor with an ED₅₀ of 12 ng/ml, indicating that theActRIII binding assay is a sensitive in vitro binding assay for GDF-8.

Example 5 Inhibition of GDF-8 and BMP-11 by GDF-8 Propeptide

When GDF-8 was preincubated with GDF-8 propeptide for 1 hour at roomtemperature, the biological activity of GDF-8 was reduced. FIG. 3 showsinduction of pGL3(CAGA)₁₂ reporter activity at the ED₅₀ for GDF-8, 10ng/ml, in the presence of GDF-8 propeptide. GDF-8 propeptide reduced theGDF-8 induction in a dose-responsive manner, with an IC₅₀ of 25 ng/ml(0.6 nM). GDF-8 propeptide also inhibited the biological activity ofBMP-11 to the same extent. In contrast, the activity of activin in thisassay was not affected by GDF-8 propeptide, presumably due to therelatively low homology between GDF-8 and activin, as compared to GDF-8and BMP-11.

Likewise, FIG. 4 shows that preincubation of GDF-8 propeptide with thebiotinylated GDF-8 at 5 ng/ml inhibited GDF-8 binding to ActRIIB in theActRIIB binding assay, as described in Example 4, with an IC₅₀ of 0.3nM. In conclusion, the GDF-8 propeptide of the invention is a potentsubnanomolar inhibitor of GDF-8 and BMP-11 activity in vitro, asmeasured in the reporter gene assay and the ActRIIB binding assay.Accordingly, this assay shows that the GDF-8 propeptide is active and isa potent inhibitor of GDF-8 activity in this assay.

Example 6 Inhibition of GDF-8-Receptor Binding by GDF-8 Propeptide

GDF-8 was iodinated with chloramine T as described by Frolik et al.(1984) J. Biol. Chem. 259:10995-10999 to a specific activity of 100-200μCi/μg. Iodinated-GDF-8 showed comparable biological activity tounlabeled GDF-8 in the (CAGA)₁₂ reporter assay described in Example 3.¹²⁵I-labeled GDF-8 was evaluated for specific binding to a myoblast cellline, L6.

L6 myoblast cells (ATCC CRL-1458) were grown to confluence in Dulbecco'smodified Eagle's medium supplemented with 10% heat-inactivated fetalcalf serum on gelatinized 24-well plates. Equilibrium binding wasperformed as described in Song et al. (1-995) Endocrinology136:4293-4297, which is incorporated by reference in its entiretyherein. 1 ng/ml ¹²⁵I GDF-8 (with or without unlabeled GDF-8) wasincubated with confluent L6 cells for 4 hours at 4° C. After washingcells, bound [¹²⁵I]GDF-8 was extracted and quantified with a gammacounter. Results are expressed as mean±S.E. of triplicate determinationsand are representative of three separate experiments.

As shown in FIG. 5, ¹²⁵I-GDF-8 bound to L6 cells with high affinity.Notably, BMP-11, but not TGF-βl, displaced ¹²⁵I-GDF-8 binding,indicating that GDF-8 and BMP-11 (but not TGF-β1) share a commonreceptor on these cells (data not shown). From a Scatchard analysis ofthe GDF-8 binding, the K_(d) was estimated to be 140 pM. Furthermore,when 1 ng/ml ¹²⁵I-GDF-8 was preincubated with GDF-8 propeptide for 1hour at room temperature, specific GDF-8 binding to L6 cells could beinhibited with increasing concentrations of unlabelled GDF-8 propeptide(FIG. 6). Results are expressed as mean±S.E. of triplicatedeterminations and are representative of two separate experiments. TheIC₅₀ for 1 ng/ml GDF-8 was 140 ng/ml GDF-8 propeptide. As evidenced bythese data, GDF-8 propeptide inhibits GDF-8 biological activity byblocking GDF-8 receptor binding.

Example 7 Inhibition of GDF-8 Activity by GDF-8 Propeptide-Fc FusionProteins

As shown in Example 8 below, murine GDF-8 propeptide has a relativelyshort in vivo half-life. To increase the bioavailability of the GDF-8propeptide, fusions between the GDF-8 propeptide and a murine IgG2a Fcregion were constructed using standard genetic engineering techniques.

Four mutations were introduced into the Fc region to reduce effectorfunction, as described in Steurer et. al. (1995) J. Immunol.155:1165-1174. Using standard PCR methodology, two fusion constructswere prepared by fusing a cDNA encoding the murine GDF-8 propeptide(amino acids 1-267) to the murine IgG2a Fc region (FIG. 7). Fusion 1(FIG. 7A) encodes the first 265 amino acids of the murine GDF-8propeptide (including a 24 amino acid secretory leader) fused in frameto 233 amino acids of the murine IgG2a Fc region starting at the firstamino acid in the hinge section. Fusion 2 (FIG. 7B) is constructed in amanner similar to Fusion 1, except that a shortglycine-serine-glycine-serine (GSGS) linker separates the GDF-8propeptide from the murine Fc region.

The two chimera were introduced into an eukaryotic expression vector,pED (Kaufman et al. (1991) Nucleic Acids Res. 19:4485-4490) andtransiently expressed in COS-1 M6 cells (Horowitz et al. (1983) Journalof Molecular and Applied Genetics 2:147-159), using Lipofectamine 2000transfection reagent (Gibco BRL, Life Technologies, Rockville, Md., USA,Cat. No. 11668-019) according to manufacturer's protocol. After 24 hoursincubation, R1CD1 (a modified DMEM/F12 serum-free culture medium) wasadded. Conditioned medium (CM) was pooled, fresh R1CD1 replaced, and CMwas harvested again 60 hours later. Conditioned media was then pooledand purified over Protein A Sepharose CL-4B (Amersham Pharmacia Biotech,Buckinghamshire, HP7 9NA, England, Cat. No. 17-07080-01) after additionof 1/10^(th) volume of 1 M Tris pH 8.0. The purified protein was elutedin 0.1 M acetic acid, 150 mM NaCl and immediately neutralized with1/20^(th) volume of 3M Tris pH 9.0. Proteins were quantitated using astandard murine IgG ELISA protocol, and assayed in the ActRIIBcompetition ELISA along with a purified human GDF-8 propeptide, asdescribed in Example 5. The results are shown in FIG. 8. The IC₅₀'s ofthe GDF-8 propeptide-Fc fusions are in the low nanomolar range and thereis no difference between fusion 1 and 2. Fusion 1 (no linker betweenGDF-8 propeptide and murine IgG2a) was selected for use in making astable CHO cell line, and tested for inhibition of GDF-8 in the RGAassay.

The GDF-8 propeptide-Fc cDNA was subcloned into the CHO expressionplasmid pHTop and transfected into CHO/A2 cells, as described in Thieset al. (2001) Growth Factors 18:251-259, which is incorporated byreference in its entirety herein. A stable cell line (PF-4/0.02) wasestablished by selecting cells in 0.02 M methotrexate. Conditionedmedium containing the GDF-8 propeptide-Fc fusion protein from a selectedline was harvested and its pH was adjusted by addition of 10% v/v 1 MTris pH 8.0 and purified over Pharmacia rProteinA Sepharose Fast Flow(Amersham Pharmacia Biotech, Buckinghamshire, HP7 9NA, England, Cat. No.17-1279-02) previously equilibrated with 50 mM Tris 150 mM NaCl pH 7.5.Fractions were eluted with 100 mM Acetic Acid, 150 mM NaCl pH 2.5 andimmediately neutralized by adding 5% v/v 3M Tris pH 9.0. Peak fractionswere pooled and loaded onto a Pharmacia Superdex 200 Size ExclusionColumn (Amersham Pharmacia Biotech, Buckinghamshire, HP7 9NA, England,Cat. No. 17-1069-01). 0.05% Tween 80 was added to the fractions toprevent aggregation. Fractions were evaluated by SDS-PAGE on Novex 10%Tricine gels (Novex, San Diego, Calif., USA, Cat. No. EC66755).

Pooled fractions were quantitated by spectrophotometry and assayed foractivity in the ActRIIB binding assay as described in Example 4, as wellas in the reporter gene assay (FIG. 9). The IC₅₀ of the purified GDF-8propeptide-Fc fusion was 1.3 nM. The data shows that the modified GDFpropeptide of the invention comprising a GDF-8 propeptide-Fc fusionprotein has retained potent inhibitory (neutralizing) activity comparedto GDF-8 propeptide.

Example 8 Pharmacokinetics

The pharmacokinetics (PK) of GDF-8 propeptide (referred to herein as“GDF8-Pro”) and GDF-8 propeptide-Fc fusion protein (referred to hereinas “GDF8-ProFc”) were evaluated in C57BI/6J mice at a dose of 0.2ug/mouse (GDF8-Pro) or 2 ug/mouse (GDF8-ProFc) after a singleintravenous administration. The animals received a mixture of unlabelledand ¹²⁵I-labeled-GDF8-Pro or GDF8-ProFc at the doses listed above andserum concentrations were determined based on I¹²⁵ radioactivity in theserum and the specific activity of the injected dose. FIG. 10 shows theserum concentration versus time curves for both the GDF8-Pro andGDF8-ProFc. Table 1 shows the PK parameters for GDF8-Pro and GDF8-ProFcafter a single intravenous administration of 2 βg/mouse. The serumconcentrations and PK parameters for the GDF8-Pro are normalized to adose of 2 βg/mouse for comparative purposes. TABLE 1 T½ Cmax ClearanceMRT V1 Vss (hrs) (ng/mL) (mL/hr) (hrs) (mL) (mL) GDF8-ProFc 232 13920.03 286 1.4 8.7 GDF-8-Pro 2.2 390 12.4 2.3 5.1 28.3T_(1/2): half life during the terminal elimination phase.C_(max): peak serum concentrationMRT: mean residence timeV₁: initial volume of distributionV_(ss): volume of distribution at steady stateNote:GDF8-Pro-PK parameters normalized to a dose of 2 μg/mouse.

As can be seen in FIG. 10, the clearance is 400-fold slower, and thehalf-life is 100-fold longer for the GDF8-ProFc as compared to theGDF8-Pro. Both the initial volume of distribution and the volume ofdistribution at steady state are approximately 3-fold greater for theGDF8-Pro compared to the GDF8-ProFc, indicating that the GDF8-Prodistributes to a larger extent outside the vascular space compared tothe GDF8-ProFc.

Although the stabilizing effect of the Fc region of an IgG molecule on aGDF propeptide is exemplified using the mouse GDF propeptide-Fc fusionprotein (i.e., both components derived from mouse), the methods of thepresent invention can be applied to any combination of GDF propeptideand IgG components, regardless of the particular precursor protein oranimal species from which the components are derived, provided thefusion protein is properly constructed to retain activity (i.e., asdescribed herein).

Example 9 Derivation and Activity of Two Human GDF-8 Propeptide FcFusions

Two GDF-8 propeptide-Fc fusion constructs were prepared, using standardPCR methodology, for evaluation of therapeutic potential. A cDNAencoding the human GDF-8 propeptide (SEQ ID NO:5) was fused to the humanIgG1 Fc region (SEQ ID NO:15; FIG. 11A) or human IgG1Fc modified forreduced effector function (SEQ ID NO:16; FIG. 11B).

1. Human GDF-8 propeptide-IgG1 wt Fc fusion protein (FIG. 11A):

The first 264 amino acids of the human GDF-8 propeptide (including aminoacids 1-241 of SEQ ID NO.: 5 and the 23-amino acid signal peptide as setforth in SEQ ID NO: 13) were fused in frame with the 232 amino acids ofthe human IgG1 constant region, starting at the first amino acid in thehinge region (SEQ ID NO.:15).

2. Human GDF-8 propeptide-IgG1 mutant (FIG. 11B):

The first 264 amino acids of the human GDF-8 propeptide, as describedabove, were fused in frame with the 227 amino acids of a mutated humanIgG1 Fc region (SEQ ID NO.:16). Two mutations, alanine residues (Ala-14and Ala-17) at set forth in SEQ ID. NO.: 16 were introduced in order toreduce effector function as described in Lund et al. (1991) J. Immun.147:2657-2662 and Morgan et al. (1995) Immunology 86:319-324.

The two fusion proteins were introduced into an eukaryotic expressionvector, pED (Kaufman et al. (1991) Nucleic Acids Res. 19:4485-4490) andtransiently expressed in COS-1 M6 cells (Horowitz et al. (1983) Journalof Molecular and Applied Genetics 2:147-159), using Lipofectamine 2000transfection reagent (Gibco BRL, Life Technologies, Rockville, Md., USA,Cat. No. 11668-019) according to manufacturer's protocol. After 24 hoursincubation, R1CD1 (a modified DMEM/F12 serum-free culture medium) wasadded. Conditioned medium (CM) was pooled, fresh R1CD1 replaced, and CMwas harvested again 60 hours later. Conditioned media was then pooledand purified over Protein A Sepharose CL-4B (Amersham Pharmacia Biotech,Buckinghamshire, HP7 9NA, England, Cat. No. 17-07080-01) after additionof 1/10^(th) volume of 1 M Tris pH 8.0. The purified protein was elutedin 0.1 M acetic acid, 150 mM NaCl and immediately neutralized with1/20^(th) volume of 3M Tris pH 9.0. Proteins were quantitated using astandard human IgG ELISA protocol, and assayed in the ActRIIB bindingassay along with a purified human GDF-8 propeptide, as described inExample 5. The results are shown in FIG. 12. In conclusion, both humanpropeptide-Fc fusion proteins are potent inhibitors of GDF-8 binding toActRIIB.

Example 10 In Vivo Testing of GDF-8 Propeptide-Fc Fusion Protein

The murine GDF-8 propeptide-Fc fusion protein (from Example 9) wastested in adult mice. Seven to nine week old, adult, female BALB/c micewere randomized with respect to body weight and placed into groups ofseven (except for the no treatment group, which had six mice). Mice weredosed twice weekly by I.P. injection with a total weekly dose of 10 ug,100 ug, or 1000 ug per animal for five weeks. Control injections weremurine IgG2a-Fc at a molar equivalent to the high dose of propeptide-Fc.At the end of the study, gastrocnemius, quadriceps, epididymal fat pad,kidney, and liver were removed and weighed. FIG. 13 shows the averagetissue mass, with the error bars indicating the standard error of themean. The asterisk (*) indicates a statistically significant difference(p<0.01 using a students T test) when compared with the mice treatedwith the control protein, IgG2aFc. Blocking GDF-8 activity in vivo byI.P. injection of GDF-8 propeptide-Fc fusion protein at 1 mg/week(treated mice) resulted in a 6.2% increase in gastrocnemius and 11.3%increase in quadricep muscle mass compared to control mice not receivingGDF-8 propeptide-Fc fusion protein. In addition, blocking GDF-8 activityin vivo by I.P. injection of GDF-8 propeptide-Fc fusion protein at 100ug/week (treated mice) results in 23.0% increase in fat pad mass. Thehigh dose (1 mg/week) treatment also led to a decrease in fat pad mass,but due to the variability in pat pad mass, the difference was nothighly statistically significant (p=0.047). There was no effect oftreatment on the mass of other tissues tested, including liver andkidney. In summary, the modified GDF-8 propeptide blocked GDF-8 activityin vivo and lead to an increase in muscle mass and a decrease in fatmass. In summary, blocking GDF-8 activity in vivo by I.P. injection ofGDF-8 propeptide-Fc fusion protein (treated mice) resulted in increasedgastrocnemious and quadricep muscle mass compared to control mice notreceiving GDF-8 propeptide-Fc fusion protein.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1-118. (canceled)
 119. A nucleic acid molecule encoding a GDF-8propeptide comprising a nucleic acid sequence that encodes a GDF-8propeptide that is at least 95% identical to SEQ ID NO:5 and that ismodified at the aspartate residue corresponding to aspartate 76 of SEQID NO:5, wherein the encoded propeptide inhibits at least one GDF-8biological activity.
 120. The nucleic acid of claim 119, wherein theGDF-8 propeptide has an increased in vivo or in vitro half life relativeto a corresponding unmodified GDF-8 propeptide.
 121. The nucleic acid ofclaim 119, wherein the GDF-8 propeptide inhibits one or more GDF-8activities chosen from negative regulation of skeletal muscle mass,modulation of preadipocyte differentiation, inhibition of muscleformation, inhibition of muscle cell growth, inhibition of muscledevelopment, regulation of muscle-specific enzymes, inhibition ofmyoblast cell proliferation, modulation of preadipocyte differentiationto adipocytes, increasing sensitivity to insulin, regulation of glucoseuptake, glucose hemostasis, and modulation of neuronal cell developmentand maintenance.
 122. The nucleic acid of claim 119, wherein the nucleicacid encodes an alanine residue at the position corresponding toaspartate 76 of SEQ ID NO:5.
 123. The nucleic acid of claim 119 furthercomprising a nucleic acid sequence encoding an Fc region of an IgGmolecule.
 124. The nucleic acid of claim 123, wherein the IgG moleculeis IgG1 or IgG4.
 125. The nucleic acid of claim 123, wherein the nucleicacid encodes an amino acid sequence that is at least 95% identical toSEQ ID NO:16.
 126. The nucleic acid of claim 119, further comprising anucleic acid sequence encoding albumin or an albumin derivative. 127.The nucleic acid of claim 119, wherein the nucleic acid encodes amodified GDF-8 propeptide with an altered glycosylation site.
 128. Thenucleic acid of claim 119, wherein the nucleic acid encodes a secondmoiety.
 129. The nucleic acid of claim 128, wherein the second moiety isa protein or a polypeptide.
 130. The nucleic acid of claim 119, whereinthe GDF-8 propeptide further comprises an immunoglobulin molecule, or afragment thereof.
 131. A nucleic acid molecule encoding a GDF-8propeptide comprising a nucleic acid sequence that is at least 95%identical to SEQ ID NO:6 and encodes a propeptide that is modified atthe aspartate residue corresponding to aspartate 76 of SEQ ID NO:5,wherein the encoded propeptide inhibits at least one GDF-8 biologicalactivity.
 132. A nucleic acid molecule encoding a GDF-8 propeptidecomprising a nucleic acid that hybridizes under stringent hybridizationconditions (hybridization at 65° C. in 6×SSC) to the complement of SEQID NO:6 and encodes a propeptide that is modified at the aspartateresidue corresponding to aspartate 76 of SEQ ID NO:5, wherein theencoded propeptide inhibits at least one GDF-8 biological activity. 133.The nucleic acid of claim 132, wherein the GDF-8 propeptide negativelyregulates skeletal muscle mass.
 134. A pharmaceutical compositioncomprising the nucleic acid of claim 119 and a pharmaceuticallyacceptable carrier or vector.
 135. A vector comprising the nucleic acidof claim
 119. 136. The vector of claim 135 further comprising a nucleicacid that encodes an Fc region of an IgG molecule.
 137. The vector ofclaim 136 further comprising a linker peptide between the nucleotidesencoding the GDF-8 propeptide and the IgG Fc region.
 138. The vector ofclaim 137, wherein the linker peptide comprises nucleotides encoding forthe amino acid sequence consisting of glycine-serine-glycine-serine(GSGS) (SEQ ID NO: 17).
 139. A host cell comprising the vector of anyone of claims 135, 136, 137 and
 138. 140. The method of making a GDF-8propeptide that has been modified at the aspartate residue correspondingto aspartate 76 of SEQ ID NO:5 comprising: (a) preparing a nucleic acidmolecule according to claim 119, 131, or 132; (b) preparing a nucleicacid molecule encoding the Fc region of the IgG molecule; and (c) fusingthe a nucleic acid molecules from steps (a) and (b) to produce amodified GDF-8 propeptide.
 141. The method of claim 140, furthercomprising preparing a double stranded oligonucleotide encoding a linkerpeptide and fusing the cDNA molecules from steps (a) and (b) to eitherend of the double stranded oligonucleotide encoding the linker peptide.142. The method of claim 141, wherein the linker peptide comprises theamino acid sequence consisting of glycine-serine-glycine-serine (GSGS)(SEQ ID NO: 17).
 143. A method of treating a patient suffering from amedical disease or disorder comprising: administering a therapeuticallyeffective amount of a GDF-8 propeptide that has been modified at theaspartate residue corresponding to aspartate 76 of SEQ ID NO:5 and apharmaceutically acceptable patient, wherein the GDF-8 propeptidecomprises an amino acid sequence that is at least 95% identical to SEQID NO:5, wherein the modification is a mutation at aspartate 76 of SEQID NO:5, and wherein the GDF-8 propeptide inhibits one or more GDF-8activities chosen from GDF-8 propeptide binding, negative regulation ofskeletal muscle mass, modulation of preadipocyte differentiation,inhibition of muscle formation, inhibition of muscle cell growth,inhibition of muscle development, regulation of muscle-specific enzymes,inhibition of myoblast cell proliferation, modulation of preadipocytedifferentiation to adipocytes, increasing sensitivity to insulin,regulation of glucose uptake, glucose hemostasis, and modulation ofneuronal cell development and maintenance thereby treating a patientsuffering from the medical disease or disorder.
 144. The method of claim143, wherein the GDF-8 propeptide has an increased in vivo or in vitrohalf life relative to a corresponding unmodified GDF-8 propeptide. 145.The method of claim 143, wherein the GDF-8 propeptide inhibits negativeregulation of skeletal muscle mass.
 146. The method of claim 143,wherein the GDF-8 propeptide further comprises an Fc region of an IgGmolecule.
 147. The method of claim 146, wherein the IgG molecule is IgG1or IgG4, or a derivative of IgG1 or IgG4.
 148. The method of claim 146,wherein the Fc region of an IgG molecule is at least 95% identical toSEQ ID NO:16.
 149. The method of claim 143, wherein the medical disorderis a muscular or neuromuscular disorder.
 150. The method of claim 149,wherein the muscular or neuromuscular disorder is muscular dystrophy.151. The method of claim 149, wherein the muscular or neuromusculardisorder is amyotrophic lateral sclerosis.
 152. The method of claim 149,wherein the muscular or neuromuscular disorder is selected from thegroup consisting of muscle atrophy, congestive obstructive pulmonarydisease, muscle wasting syndrome, sarcopenia, and cachexia.
 153. Themethod of claim 143, wherein the medical disorder is a metabolicdisorder.
 154. The method of claim 153, wherein the metabolic disorderis selected from the group consisting of type 2 diabetes,noninsulin-dependent diabetes mellitus, hyperglycemia, and obesity. 155.The method of claim 143, wherein the medical disorder is a bonedegenerative disorder.
 156. The method of claim 155, wherein the bonedegenerative disorder is osteoporosis.
 157. The method of claim 143,wherein the medical disorder is an adipose tissue disorder.